Insulin action to recruit an intracellular pool of the glucose transporter protein GLUT4 to the fat/muscle cell surface has been established for more than a decade, yet the molecular details of this phenomenon are still elusive. Czech, M. P., Ann. Rev. Nutr. 15:441-471 (1995). Intriguingly, while GLUT4 appears to be a unique isoform for fat and muscle tissues, signaling element(s) specifically implicated in its sorting, directing and insulin-sensitive delivery to the cell surface are presently unknown. Phosphatidylinositol4-phosphate-5-kinase (PIP5K; EC2.7.1.68) has been implicated in membrane trafficking in yeast. Yamamoto, A. et al., Mol. Biol. Cell. 6:525-539 (1995). In particular, PIP5K synthesizes phosphatidylinositol 4,5-bisphosphate (Ptdlns[4,5]P.sub.2) from phosphatidylinositol-4-phosphate (Ptdlns[4]P). Loijens, J. C. et al., Advan. Enzyme Regul. 36:115-140 (1996). It has recently been reported that PIP5K also synthesizes Ptdlns[4,5]P.sub.2 from phosphatidylinositol-5-phosphate (Ptdlns[5]P). Hinchliffe, K. et al., Nature 390:123-124 (1997). The biosynthesis of Ptdlns[4,5]P.sub.2 has attracted increasing interest because of mounting evidence implicating metabolites of Ptdlns[4,5]P.sub.2 as important regulators of many cellular processes. Loijens, J. C. et al., Advan. Enzyme Regul. 36:115-140 (1996). In particular, Ptdlns[4,5]P.sub.2 is a key substrate of insulin-activated Pl 3-kinase, which enzyme, together with its Ptdlns[3,4]P.sub.2 and Ptdlns[3,4,5]P.sub.3 products, appear to be important elements in insulin action on GLUT4 membrane movements. Czech, M. P., Ann. Rev. Nutr. 15:441-471 (1995).
The key role of activated Pl 3-kinase implies the presence of a large, easily available phosphoinositide substrate pool and suggests that the local production of Ptdlns[4]P and Ptdlns[4,5]P.sub.2 lipid substrates at key insulin-sensitive intracellular locations would aid an efficient Pl 3-kinase reaction and may be crucial for the Pl 3- kinase-mediated effect of insulin in GLUT4 directing and delivery to the fat/muscle cell surface. In addition, an alternative pathway of generating Ptdlns[3,4]P.sub.2 and Ptdlns[3,4,5]P.sub.3 has been recently suggested which utilizes Ptdlns[3]P substrates and concert action of phosphatidylinositol-4-phosphate-5-kinases. Zhang, X. et al., J. Biol. Chem. 272:17756-17761 (1997). Taken together, these data are consistent with the notion that the activity of PIP5K can contribute to the regulated pools of Ptdlns[3,4]P.sub.2 and Ptdlns[3,4,5]P.sub.3 stimulated by growth factors and insulin.
Two distinct mammalian PlP5Ks, called type I (PIP5KI) and type II (PIP5KII), isolated from bovine and human erythrocytes, respectively, have been reported (Bazenet, C. E. et al., J. Biol. Chem. 265: 18012-18022 (1990); Jenkins, G. H. et al., J. Biol. Chem. 269:11547-11554 (1994)), as well as an isoform of PIP5KII (PIP5KIIa). Boronenkov, I. V. et al., J. Biol. Chem. 270:2881-2884 (1995). Yeast isozymes, specifically MSS4 and fab1, have also been isolated and studied. Yamamoto, A. et al., Mol. Biol. Cell 6:525-539 (1995); Yoshida, S. et al., Mol. Gen. Genet. 342:631-640 (1994); Yamamoto, A. et al., Mol. Biol. Cell 6:525-539 (1995).
As mentioned above, the conversion from Ptdlns[4]P to Ptdlns[4,5]P.sub.2 is an important branchpoint in the phosphoinositide (PI) cycle, depicted in FIG. 1A. FIG. 1B depicts newly described inositol lipids, Ptdlns[5]P and Ptdlns[3,5]P.sub.2, and FIG. 1C includes the novel alternative pathway for Ptdlns[3,4,5]P.sub.3 production by PIP5Ks. The hydrolysis of Ptdlns[4,5]P.sub.2 by phosphoinositide-specific phospholipase C(PLC; EC 3.1.4.3) generates the second messengers, 1,2-diacylglycerol and inositol 1 ,4,5-triphosphate. 1,2-diacylglycerol activates several protein kinase C isoforms while inositol 1,4,5-triphosphate causes an increase in intracellular calcium. Rana, R. S. Physiol. Rev. 70:115-164 (1990). Ptdlns[4,5]P.sub.2 can also be phosphorylated by a PI 3-kinase (EC 2.7.1.137) to phosphatidylinositol 3,4,5-triphosphate (Ptdlns[3,4,5]P.sub.3), a second messenger whose targets are largely unknown but may include protein kinase C isoforms. Nakanishi, H. et al., J. Biol. Chem. 268:13-16 (1993); Toker, A. et al., J. Biol. Chem. 269:32358-32367 (1994). Furthermore, Ptdlns[4,5]P.sub.2 modulates the function of numerous enzymes including many actin-binding proteins (Janmey, P. A., Annu. Rev. Physiol. 56:169-191 (1994)), binds Ph domains found in some signaling proteins (Harlan, J. E. et al., Nature 371:168-170 (1994)), and appears to be involved in the secretory vesicle cycle. Eberhard, D. A. et al., Biochem. J. 268:15-25 (1990); Hay, J. C. et al., Nature 374:173-177 (1995); Liscovitch M. et al., Cell 81:659-662 (1995).
PIP5Ks have been isolated from erythrocytes, brain, adrenal medulla, liver and other sources. Carpenter, C. L. et al., Biochemistry 29:11147-11156 (1990) (and references therein); Van Dongen, C. J. et al., Biochem. J. 233:859-864 (1986); Moritz, A et al., Biochim. Biophys. Acta 1168:79-86 (1993); Divecha, N. et al., Biochem. J. 288:637-642 (1992); Husebye, E. S. et al., Biochim. Biophys. Acta 1010:250-257 (1989); Urumow, T. et al., Biochim. Biophys. Acta 1052:152-158 (1990). In cells, PIP5K activity is found on the plasma membrane (Carpenter, C. L. et al., Biochemistry 29:11147-11156 (1990); Urumow, T. et al., Biochim. Biophys. Acta 1052:152-158 (1990); Ling, L. E. et al., J. Biol. Chem. 264:5080-5088 (1989); Smith, C. D. et al., J. Biol. Chem. 264:3206-3210 (1989); Bazenet, C. E. et al., J. Biol. Chem. 265:18012-18022 (1990); Jenkins, G. H. et al., J. Biol. Chem. 269:11547-11554 (1994)), associated with the cytoskeleton (Payrastre, B. et al., J. Cell Biol. 115:121-128 (1991); Grondin, P. et al., J. Biol. Chem. 266:15705-15709 (1991)), on the endoplasmic reticulum (Helms, J. B. et al., J. Biol. Chem. 266:21368-21374 (1991), and in nuclei (Divecha, N. et al., Biochem. J. 289:617-620 (1993); Payrastre, B. et al., J. Biol. Chem. 267:5078-5084 (1992); Divecha, N. et al., Cell 74:405-407 (1993)). There is also a soluble, cytosolic population of PIP5K. Ling, L. E. et al., J. Biol. Chem. 264:5080-5088 (1989); Bazenet, C. E. et al., J. Biol. Chem. 265:18012-18022 (1990); Jenkins, G. H. et al., J. Biol. Chem. 269:11547-11554 (1994); Moritz, A. et al., J. Neurochem. 54:351-354 (1990). The kinase's product, Ptdlns[4,5]P.sub.2, is primarily found in the plasma membrane but can be detected in isolated endoplasmic reticulum and nuclei. Helms, J. B. et al., J. Biol. Chem. 266:21368-21374 (1991); Tran, D. et al., Cell. Signal 5:565-581 (1993). Ptdlns[4]P is present in all of these fractions.
Helms, J. B. et al., J. Biol. Chem. 266:21368-21374 (1991); Tran, D. et al., Cell. Signal 5:565-581 (1993). Hinchliffe, K. et al., Nature 390:123-124 (1997); Rameh, L. E. et al., Nature 390:192-196 (1997).
One postulated reason for the large family of PIP5Ks is that many forms of regulation and cellular functions have been attributed to PIP5Ks, as summarized in FIG. 2. It would thus be desirable to provide a mechanism to further study the role of PIP5Ks. It would also be desirable to provide a novel mammalian PIP5K. It would further be desirable to provide a screening method for further studying the role of PIP5Ks, their substrates and products. It would still further be desirable to provide an animal model for further investigating the role of PlP5Ks.